ESTANDARIZACIÓN

The incubation study has demonstrated that the higher urine application rate of 700 kg N ha-1 significantly increased the main effects for the soil NH4+-N concentration (p < 0.001) and AOB

abundance (p = 0.035) compared to urine being applied at 500 kg N ha-1. However, it significantly (p = 0.03) decreased the main effect of the AOA abundance and there was no statistical difference in soil NO3--N concentrations.

0 20 40 60 80 100 120 140 160 0 10 20 30 40 50 60 70 80 NO 3 --N c o n ce n tr ati o n ( mg N O3 --N kg -1 d ry s o il)

AOB amoA gene copy numbers (million copies g-1 _{dry soil)}

(a) 0 20 40 60 80 100 120 140 160 0 1 2 3 4 5 NO 3 - -N c o n ce n tr ati o n (mg N O3 - -N kg -1 d ry so il)

AOA amoA gene copy numbers (million copies g-1 _{dry soil)}

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As the cow urine-N concentration increases it directly impacts on the nitrification rate due to there being a greater amount of NH3 in the soil. Increasing the rate of nitrification subsequently

increases the AOB amoA gene abundance in response to the greater amount of NH3 present. The

main effect of NH4+-N concentration increased by 38% and the main effect of the AOB amoA

gene abundance increased by 19% when cow urine was applied at 700 kg N ha-1 compared to 500 kg N ha-1. Subsequently, higher cow urine-N concentrations could potentially increase NO3--N leaching losses. Di and Cameron (2007) supports the idea of an increasing urine-N

concentration increases NO3--N leaching losses. They reported that there was a significant

difference in NO3--N leaching losses when animal urine-N application rate increased from 300 kg

N ha-1 to 700 kg N ha-1 to 1000 kg N ha-1 due to increasing nitrification rates.

However, there was no significant difference observed between the main effect NO3--N

concentration between urine applied at 700 kg N ha-1 compared to 500 kg N ha-1.Conversely, by day 112, a significant difference in soil NO3--N concentration occurred as the result of different

nitrification rates (Figure 6.4). The nitrification rate for the urine 500 treatment had decreased by this time as the result of low NH4+-N levels, thus low soil NO3--N concentrations resulted. The

urine 700 treatment still had sufficient NH4+-N for nitrification, thus higher soil NO3--N resulted. If

the trial continued for a longer period of time there would have likely been a difference in peak NO3--N concentrations between the two urine application rates, thus further supporting the idea

that higher cow urine-N rates increase NO3- leaching losses.

6.4.2 The roles AOB and AOA

This study confirms the findings reported in Chapters Four and Five - that AOB mediated the nitrification process under dairy winter forage grazing conditions. When cow urine was applied to the soil significant AOB population growth followed. The urine application rates of 500 kg N ha-1 and 700 kg N ha-1 caused the AOB amoA gene abundance to increase by 8.1-fold and 7.8-fold respectively (Figures 6.7). Similarly, on day 56, a significant increase in AOB amoA

transcript abundance was observed when cow urine was applied to the soil (Figure 6.13a). These findings are similar to that of Di et al. (2009b) who showed that AOB mediated nitrification under high N loading. The greater AOB population growth resulted from higher nitrification rates (Kowalchuk & Stephen 2001; Di et al. 2010) which was a direct response to the addition of urine that produced NH3. The treatments with DCD present had lower AOB populations due to DCD

inhibiting AOB growth (Amberger 1989; McCarty & Bremner 1989; Di et al. 2009a). A significant (p < 0.001) positive relationship was determined between the AOB population and soil

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nitrification rates (Figure 6.14a), thus further supporting the link between AOB populations and soil nitrification rates under high N loading.

In contrast, no relationship was identified between the AOA population and soil nitrification rates (Figure 6.14b), suggesting that AOA are not related to the rate of nitrification under dairy winter forage grazing conditions. Conversely, a significant (p = 0.002) decrease in AOA population growth was observed when cow urine at 700 kg N ha-1 was applied to the soil compared to no urine being applied (Figure 6.10). This is in agreement with previous studies (Di et al. 2009b; Jia & Conrad 2009; Di et al. 2010). In addition, the application of urine at 700 kg N ha-1 showed a significant (p = 0.030) reduction in AOA abundance compared to the lower urine application rate of 500 kg N ha-1 (Figure 6.10), suggesting that a higher application rate of cow urine-N suppresses the growth of AOA. Di et al. (2009b) and Di et al.(2010) previously suggested that AOA population growth was inhibited as the soil NH3 concentration increased. From this, Di

et al.(2010) hypothesised that AOA prefer different growing conditions to that of AOB - with AOB preferring high NH3 substrate conditions while AOA prefer lower NH3 substrate conditions.

The current study also identified a significantly (p < 0.001) greater AOB abundance compared to the AOA abundance. This finding supports the results of Chapter Five. However, it is in contrast to the findings of Leininger et al. (2006) and He et al. (2007) who reported that AOA was the numerically dominant ammonia oxidiser. This difference in ammonia oxidiser abundance was most likely due to the high N loading in the current study which is the preferred growing conditions for AOB rather than AOA.

6.4.3 Effect of DCD

The incubation study confirms that the use of DCD effectively reduced NO3- accumulation within

a soil by inhibiting nitrification under simulated dairy winter forage grazing conditions. This is similar to that of pervious work under several agricultural systems (Williamson et al. 1996; Di & Cameron 2002b, 2004a; Di et al. 2009a; Moir et al. 2010) and the findings in Chapters Four and Five. The application of DCD was highly effective in inhibiting the growth and activity of AOB populations and therefore reduced the rate of nitrification (Figures 6.8a, b, and 6.13a). This led to a significant (p < 0.001) reduction in the soil NO3--N concentration for the duration of the

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It has previously been determined that environmental factors and DCD movement through the soil profile affect the effectiveness of DCD (Amberger 1989; McCarty & Bremner 1989; Kelliher et al. 2008; Shi et al. 2011; Shepherd et al. 2012a). With a soil temperature of 10oC, this incubation study has shown that DCD can remain in the soil and be effective for the 112 days. The soils on dairy winter grazing forage systems are wet and anaerobic, in addition to having a low soil temperature, which could possibly slow down the rate of DCD degradation, increasing the effectiveness of DCD. However, leaching can also remove DCD from the soil thus decreasing its effectiveness.

The incubation study showed that both DCD application rates of 10 kg DCD ha-1 and 20 kg DCD ha-1 were effective in reducing soil nitrification rates under high N loading by significantly (p < 0.001) reducing the NO3--N concentrations, AOB amoA gene abundance, and AOB amoA

transcript abundance. This is similar to that of Di and Cameron (2004b) who showed that the DCD application rates of 7.5 kg DCD ha-1 and 15 kg DCD ha-1 inhibited the rate of nitrification. This study went on to show that except for the soil NO3--N concentration (p < 0.001) there were

no statistical differences between the two DCD application rates. Since NO3- concentration is the

most important parameter affecting NO3- leaching, it should be recommended to farmers that

one 20 kg DCD ha-1 application of DCD be applied to dairy winter forage soil to maximise the environmental benefits of deceasing NO3- leaching. However, more research is required on the

effect of different application rates to verify the results.

6.4.4 The use of biochar

Biochar did not mitigate NO3--N accumulation in the soil, supporting the conclusions of Chapter

Four. However, due to biochar having the ability to enhance NH3, NH4+, and NO3- retention,

biochar has previously shown to reduce soil NO3- concentrations (Steiner et al. 2008; Knowles et

al. 2011; Dempster et al. 2012a; Yao et al. 2012). Thus, why has the current study shown no reductions in soil NO3--N concentrations with the addition of biochar? One reason could be the

rate at which the biochar was applied. In the current study, the application rate of biochar was at 1.75 tonnes biochar ha-1. This is low compared to the application rates of 25 tonnes biochar ha-1 and 100 tonnes biochar ha-1 used by Dempster et al. (2012a) and Knowles et al.(2011) who identified biochar reduced NO3--N leaching. The biochar was applied at a lower rate in the

current study for economic reasons, as high rates of application may be too costly and impractical for commercial use.

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In addition to the application rate of biochar affecting NO3- leaching losses, a pyrolysis

temperature above 600oC has been determined to impact on biochar’s ability to adsorb NO3- and

subsequently reduce NO3--N leaching (Dempster et al. 2012a; Yao et al. 2012; Clough et al.

2013). Thus, due to the pyrolysis temperature being 550oC in the current study, it is unlikely that biochar adsorption of NO3- occurred.

Unlike Chapter Four, where biochar was applied to the soil’s surface, the biochar was mixed into the soil in the incubation study. Biochar was mixed into the soil as this method was shown to be most effective at reducing NO3--N leaching (Knowles et al. 2011; Dempster et al. 2012a).

However, Dempster et al.(2012a) and the current study had different initial biochar materials. Dempster et al.(2012a) used Jarrah wood (Eucalyptus marginata)pyrolysed at 600oC whereas the current study used Pinus radiata pyrolysed at 550oC. However, it is unlikely that this difference in source material could account for the NO3--N accumulation differences as Knowles

et al. (2011) also used Pinus radiata yet reported similar results to Dempster et al. (2012a). Therefore, the effect of biochar on NO3- accumulation in soil may vary depending on the rates of

application and possibly other conditions.

6.5 Conclusions

The higher cow urine application rate of 700 kg N ha-1 resulted in a greater nitrification rate compared to the application rate of 500 kg N ha-1. The application of DCD at either 10 kg DCD ha-1 or 20 kg DCD ha-1 significantly reduced the nitrification rate thus making DCD a suitable mitigation tool against NO3- leaching under dairy winter forage conditions. However, biochar at

the rate used in this study was not effective in reducing the rate of nitrification under the same conditions. The AOB population was more abundant than the AOA population and was the dominant ammonia oxidiser. Thus, when DCD is applied it is the inhibition of AOB that decreases soil NO3--N concentrations under dairy winter forage grazing condition.

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Chapter 7

Conclusions

7.1 Introduction

A review of the literature identified clear gaps in the research regarding NO3- leaching losses and

mitigation on dairy winter forage grazing systems. The research described in this thesis has addressed these knowledge gaps.